Cell-type-resolved Metabolic Flux Inference Reveals Stromal Metabolic Reprogramming Across Human Cardiomyopathies

Metabolic remodeling is a hallmark of cardiomyopathy, yet which cell types bear the metabolic burden and how cell-type-specific contributions are disrupted remain unclear. Here, we developed a cell-type-resolved genome-scale metabolic flux inference pipeline optimized for post-mitotic cardiac tissue by maximizing ATP synthesis rather than biomass production and applied it to a single-nucleus transcriptomic atlas of human cardiomyopathies (78 donors, 869,449 nuclei). Metabolic impairment in dilated cardiomyopathy (DCM) was most profound in stromal cells, whereas myeloid cells exhibited opposing metabolic activation. DCM- associated impairment followed a genotype-dependent severity gradient from structural gene mutations to pathogenic variant-negative (PVneg) cases. PVneg hearts uniquely harbored 24 altered metabolic pathways not significant in any other genotype. These PVneg-specific signatures were independent of clinical severity, indicating a genotype-intrinsic metabolic program. Extending the analysis to arrhythmogenic cardiomyopathy and hypertrophic cardiomyopathy showed that ATP depletion is shared across cardiomyopathy subtypes, whereas metabolic remodeling differed across disease subtypes. Additionally, gene regulatory network analysis linked these alterations to broad transcription factor (TF) dysregulation and pervasive TF-metabolic coupling across all cell types. These findings redefine PVneg DCM as a metabolically distinct entity and reveal conserved stromal metabolic remodeling across cardiomyopathies, providing a framework for genotype-informed mechanistic stratification.